-
The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates
adefense-related NAC transcription factor and is involved indefense
signaling
Min Miao1,2*, Xiangli Niu1,3*, Joanna Kud2*, Xinran Du2, Julian
Avila4, Timothy P. Devarenne4,Joseph C. Kuhl2, Yongsheng Liu1,3 and
Fangming Xiao2
1School of Biotechnology and Food Engineering, Hefei University
of Technology, Hefei 230009, China; 2Department of Plant, Soil and
Entomological Sciences, University of Idaho, Moscow
ID 83844-2339, USA; 3Ministry of Education Key Laboratory for
Bio-resource and Eco-environment, College of Life Science, State
Key Laboratory of Hydraulics and Mountain River
Engineering, Sichuan University, Chengdu, Sichuan 610064, China;
4Department of Biochemistry and Biophysics, Texas A&M
University, College Station, TX 83844-2339, USA
Author for correspondence:Yongsheng LiuTel: +86 13808030238
Email: [email protected]
Fangming Xiao
Tel: +1 785 885 0120
Email: [email protected]
Received: 26 October 2015Accepted: 10 January 2016
New Phytologist (2016) 211: 138–148doi: 10.1111/nph.13890
Key words: NAC (NAM, ATAF1,2, CUC2)transcription factor, plant
defense response,SEVEN IN ABSENTIA (SINA) ubiquitin ligase,tomato
(Solanum lycopersicum),ubiquitin–proteasome system
(UPS)-mediateddegradation.
Summary
� We recently identified a defense-related tomato (Solanum
lycopersicum) NAC (NAM,ATAF1,2, CUC2) transcription factor, NAC1,
that is subjected to ubiquitin–proteasome sys-tem-dependent
degradation in plant cells. In this study, we identified a tomato
ubiquitin ligase
(termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1,
promoting its degradation.� We conducted coimmunoprecipitation and
bimolecular fluorescence complementation todetermine that SINA3
specifically interacts with the NAC1 transcription factor in the
nucleus.
Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and
promotes NAC1 degrada-
tion via polyubiquitination in vivo, indicating that SINA3 is a
ubiquitin ligase that ubiquitinates
NAC1, promoting its degradation. Our real-time PCR analysis
indicated that, in contrast to
our previous finding that NAC1 mRNA abundance increases upon
Pseudomonas infection,
the SINA3mRNA abundance decreases in response to Pseudomonas
infection.� Moreover, using Agrobacterium-mediated transient
expression, we found that overexpres-sion of SINA3 interferes with
the hypersensitive response cell death triggered by multiple
plant
resistance proteins.� These results suggest that SINA3
ubiquitinates a defense-related NAC transcription factorfor
degradation and plays a negative role in defense signaling.
Introduction
NAC (NAM, ATAF1,2, CUC2) transcription factors belong toone of
the largest families of plant-specific transcription factorsand
consist of a conserved N-terminal NAC domain involved inDNA binding
and a highly variable C-terminal domain responsi-ble for
transcriptional activation (Puranik et al., 2012). AlthoughNAC
transcription factors were originally identified as a result
oftheir role in developmental processes, multiple lines of
evidencehave shown that they are also largely involved in plant
defenseresponse. In both the model plant Arabidopsis thaliana and
cropplants such as rice, pepper, wheat, potato and rapeseed,
numerousNAC genes are induced in response to pathogen
infection(Puranik et al., 2012). Genetic impairing or
overexpression of cer-tain NAC genes resulted in attenuated or
enhanced resistance topathogens (Delessert et al., 2005; Wang et
al., 2009; Wu et al.,
2009; Huang et al., 2013), suggesting that NAC transcription
fac-tors could positively or negatively regulate plant defense
response.
The ubiquitin–proteasome system (UPS)-mediated
proteindegradation is an important post-translational regulatory
mecha-nism and plays a significant role in many physiological
processesby removal of intracellular proteins (Harper &
Schulman, 2006;Ravid & Hochstrasser, 2008). The UPS pathway
contains E1(ubiquitin-activating), E2 (ubiquitin-conjugating) and
E3 (ubiq-uitin ligase) enzymes that function in concert to
covalently linkubiquitin to the substrate protein (Harper &
Schulman, 2006).The ubiquitin moieties are consecutively added to a
lysine residueof the substrate protein through linkages at one of
seven lysineresidues (K48, in most cases) in each ubiquitin to form
a polyu-biquitin chain, which will consequently be recognized by
the 26Sproteasome for proteolytic degradation (Harper &
Schulman,2006). The ubiquitin E1s and E2s are relatively
conserved,whereas the E3s are highly diverse and determine the
substratespecificity (Harper & Schulman, 2006). In plants,
UPS-mediated*These authors contributed equally to this work.
138 New Phytologist (2016) 211: 138–148 � 2016 The AuthorsNew
Phytologist� 2016 New Phytologist Trustwww.newphytologist.com
Research
-
degradation not only cleans up misfolded and/or damaged
pro-teins to avoid potential toxicity, but also adjusts the amount
ofregulatory proteins, in particular transcription factors and
signal-ing kinases, which allows for tight control of many
physiologicalprocesses such as senescence, development, stress
tolerance anddefense response in a temporally and spatially
specific mannar(Sadanandom et al., 2012).
The SEVEN IN ABSENTIA (SINA) ubiquitin ligase was
firstidentified in Drosophila melanogaster and is required for
forma-tion of the R7 photoreceptor involved in eye
development(Carthew & Rubin, 1990). The SINA ubiquitin ligase
contains aconserved N-terminal cysteine-rich C3H4 RING domain,
twozinc finger motifs and a C-terminal domain responsible for
sub-strate-binding and dimerization (Hu & Fearon, 1999). It
isgenerally thought that, like many other monomeric RING-containing
ubiquitin ligases, SINA and SINA homologs (Siah)directly interact
with their substrates through the substrate-binding domain (SBD)
and facilitate the ubiquitination via theRING domain (House et al.,
2003). In plants, despite the factthat a genome-wide analysis
suggests the existence of SINA ubiq-uitin ligases in many plant
species (Wang et al., 2008), only fiveSINA ubiquitin ligases have
been identified and their functionsare not fully understood (Xie et
al., 2002; Welsch et al., 2007;Den Herder et al., 2008, 2012; Park
et al., 2010; Ning et al.,2011a). In Arabidopsis, one SINA
ubiquitin ligase, SINAT5,plays roles in both lateral root growth
and floral development(Xie et al., 2002; Park et al., 2010),
whereas another, SINAT2, isinvolved in carotenogenesis (Welsch et
al., 2007). In rice, theSINA ubiquitin ligase OsDIS1 functions as a
negative regulatorin drought stress response (Ning et al., 2011b).
In Lotusjaponicas, the Lotus SINA4 negatively regulates
Sinorhizobiummeliloti infection (Den Herder et al., 2012). Finally,
in Medicagotruncatula, heterogeneous overexpression of Arabidopsis
SINAT5affects nodulation (Den Herder et al., 2008).
Although a few SINA-interacting proteins have been identifiedas
putative ubiquitinating substrates in plants, including
theArabidopsis transcription factors AtNAC1 (Xie et al., 2002),
LHY(Park et al., 2010), and RAP2.2 (Welsch et al., 2007), the
Lotussymbiosis receptor-like kinase SYMRK (Den Herder et al.,
2012)and the rice tubulin complex-related serine-threonine
proteinkinase OsNek6 (Ning et al., 2011a), only the Arabidopsis
AtNAC1and LHY have been shown to be ubiquitinated by SINAT5 (Xieet
al., 2002; Park et al., 2010). Nevertheless, these studies
suggestthat plant SINA ubiquitin ligases may regulate physiological
andcellular processes through targeting transcription factors
and/or sig-naling kinases for promoting their degradation.
We recently demonstrated that the defense-related tomato(Solanum
lycopersicum) NAC transcription factor NAC1 (Selthet al., 2005) is
fine-tuned at both transcriptional andpost-translational levels
(Huang et al., 2013). The tomato NAC1transcription factor was
originally identified as a host proteininteracting with the
geminivirus replication enhance (REn) pro-tein of tomato leaf curl
virus in a yeast two-hybrid (Y2H) screenand the relevance of this
interaction is unclear (Selth et al., 2005).A role for tomato NAC1
in the defense response is supported byour recent findings of rapid
induction of NAC1 gene expression
in tomato during Pseudomonas infection and enhanced
suscepti-bility to Pseudomonas infection in Nicotiana benthamiana
whoseNAC1 homologs were silenced (Huang et al., 2013).
Further-more, we found that the tomato NAC1 is ubiquitinated
anddegraded by the UPS in plant cells (Huang et al., 2013). Given
asignificant role of NAC1 in the defense response, we have
specu-lated that tomato plants have evolved a mechanism to
fine-tunethe abundance of NAC1 transcription factor. Under normal
con-ditions without pathogen challenge, the NAC1 gene is
expressedat a basal level and the encoded NAC1 protein is
rapidlydegraded by the UPS to prevent autoactivation of stress
responsesignaling. Upon Pseudomonas infection, the expression of
theNAC1 gene is rapidly up-regulated to produce more NAC1
tran-scription factor to compensate for its degradation, thereby
acti-vating downstream defense-related genes (Huang et al.,
2013).
In this study, we identified a tomato SINA ubiquitin ligaseSINA3
that targets NAC1 for ubiquitination and degradation. Incontrast to
up-regulation of the NAC1 gene, the expression of theSINA3 gene is
down-regulated during defense response toPseudomonas infection.
Moreover, overexpression of SINA3interferes with hypersensitive
response (HR), a localized defense-related cell death, triggered by
multiple R proteins. Together, ourexperimental data suggest that
SINA3 targets the defense-relatedNAC1 transcription factor for
degradation and plays a negativerole in defense response.
Materials and Methods
Yeast two-hybrid assay
A LexA Y2H system was used to test protein interactions
(Golemiset al., 2001). The N-terminal 260 amino acids of NAC1
(NAC11-260), which did not exhibit autoactivation, were cloned into
thebait vector pEG202 at the EcoRI and SalI sites, whereas
theSINA1-6 (the National Center for Biotechnology
Information’saccession numbers for tomato SINA1-6 genes are
AK324518,BT013026, AK322153, AK320390, AK321160 and XM_004248034)
were cloned into the prey vector pJG4-5 at EcoRI and XhoIsites,
respectively. The resulting bait and prey constructs wereintroduced
into yeast (Saccharomyces cerevisiae) EGY48 and trans-formed yeast
cells were streaked onto X-Gal plates to assess theinteractions
between NAC1 and SINA1-6. Photographs weretaken at 2 d after
incubation at 30°C. Primers used in generatingthe Y2H constructs
are listed in Supporting Information Table S1.
In vitro ubiquitination assay
SINA3 and hemagglutinin (HA) epitope-tagged NAC1-HA
werePCR-amplified from tomato leaf cDNA and pTEX::NAC1-HA(Huang et
al., 2013), respectively, and cloned into the pMAL-C2vector (NEB,
USA) at EcoRI and SalI to generate the maltosebinding protein
(MBP)-fusion proteins. The resulting constructswere introduced into
Escherichia coli BL21 where the recombi-nant proteins were
expressed with the presence of 0.5 lMisopropyl
b-D-1-thiogalactopyranoside. The in vitro ubiquitina-tion assay was
performed as described previously (Abramovitch
� 2016 The AuthorsNew Phytologist� 2016 New Phytologist
Trust
New Phytologist (2016) 211: 138–148www.newphytologist.com
NewPhytologist Research 139
-
et al., 2006) with minor modifications. The ubiquitination
reac-tion mixture (30 ll) contained 40 ng GST-E1 (AtUBA1), 100
ngGST-E2 (AtUBC8), 1 lg MBP-SINA3, 2 lg FLAG-Ub (BostonBiochem,
Cambridge, MA, USA) in the ubiquitination buffer(50 mM Tris HCl, pH
7.5, 2 mM ATP, 5 mM MgCl2, 30 mMcreatine phosphate (Sigma-Aldrich)
and 50 ng ll�1 creatinephosphokinase (Sigma-Aldrich)). The reaction
mixture was incu-bated at 30°C for 2 h and terminated by sodium
dodecyl sulfate(SDS) sample buffer. Proteins were separated with
7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
identifiedby western blotting using the a-FLAG (Sigma-Aldrich)
antibody.
The in vitro ubiquitination of NAC1 by SINA3 was per-formed
using 500 ng MBP-NAC1-HA as substrate. After incuba-tion, 15 ll
a-HA affinity matrix (Roche Applied Science,Indianapolis, IN, USA)
was added to the incubation mixture toimmunoprecipitate the
ubiquitinated MBP-NAC1-HA protein.After washing three times with
the washing buffer (20 mM TrisHCl, pH 7.5, 0.1 M NaCl, 0.1 mM EDTA,
0.05% Tween 20),proteins were separated by 7.5% SDS-PAGE and the
ubiquiti-nated NAC1-HA was determined by western blotting using
thea-FLAG or a-HA antibody (Sigma-Aldrich). Primers used
ingeneration of the MBP fusion constructs are listed in Table
S1.
Agrobacterium-mediated transient expression and
coim-munoprecipitation
Agrobacterium-mediated transient expression was carried out
asdescribed previously (Xiao et al., 2007), with the minor
modifica-tion that freshly transformed Agrobacterium tumefaciens
GV2260and relatively young plant leaves with a light green color
were usedfor agrobacterial injection. The Rpi-blb1D475V-HA and
RxD460V-HA constructs were generated in the same way as
PrfD1416V-HA,as described in our previous publication (Du et al.,
2012). For thecoIP assay, Agrobacterium-infected N. benthamiana
(Domin) leaftissues were collected at 48 h after infiltration and
ground to a finepowder with liquid nitrogen. The lysate was
resuspended in 1.0 mlprotein extraction buffer (50 mM Tris-HCl pH
7.5, 150 mMNaCl, 5 mM EDTA, 2 mM dithiothreitol (DTT), 10%
glycerol,1% polyvinylpolypyrolidone, 1 mM phenylmethylsulfonyl
fluo-ride (PMSF), plant protease inhibitor cocktail
(Sigma-Aldrich))and centrifuged at 12 000 g/4°C for 20 min. The
supernatant wasincubated with 15 ll a-HA affinity matrix (Roche
AppliedSciences) at 4°C for 2 h to capture the epitope-tagged
protein.After washing four times with washing buffer (50 mM
Tris-HCl,pH 7.5, 250 mM NaCl, 5 mM EDTA, 10% glycerol, 1 mMPMSF),
the immunoprecipitated protein complex was separatedby SDS-PAGE and
then subjected to western blotting analysisusing the a-HA or a-FLAG
antibody. Primers used in generatingthe relevant construct are
listed in Table S1.
Bimolecular fluorescence complementation (BiFC) analysis
NAC1 was PCR-amplified from tomato leaf cDNA (seeTable S1 for
primers used) and cloned into the pBSPYNE vectorcontaining the
N-terminal 155 amino acids of yellow fluores-cence protein (YFP)
protein to generate NAC1-NYFP, whereas
SINA3 or SINA31-181 was cloned into the pBSPYCE vector
con-taining the C-terminal 83 amino acids of YPF to
generateSINA3-CYFP and SINA31-181-CYFP (Walter et al.,
2004),respectively. BiFC assay was carried out via the
Agrobacterium-mediated transient expression of NAC1-NYFP with
SINA3-CYFP or SINA31-181-CYFP in N. benthamiana leaves asdescribed
previously (Walter et al., 2004). Two 4-wk-oldN. benthamiana leaves
were injected with Agrobacterium GV2260strains containing
individual BiFC construct pairs and a binaryplasmid expressing the
p19 protein to suppress gene silencing.After
40,6-diamidino-2-phenylindole (DAPI) staining, the epi-dermal cell
layers were examined using confocal microscope tosimultaneously
capture DAPI and YFP signals.
Results
The tomato defense-related transcription factor NAC1interacts
with the ubiquitin ligase SINA3
We recently characterized the involvement of the tomato
tran-scription factor NAC1 in the plant defense response and
itsUPS-mediated degradation (Huang et al., 2013). However,
thecorresponding ubiquitin ligase was not identified at that
time.Previous research demonstrated that the Arabidopsis
SINAT5protein, a RING-finger type ubiquitin ligase homologous to
theDrosophila SEVEN IN ABSENTIA (SINA) ubiquitin ligase,interacts
with and ubiquitinates the Arabidopsis NAC transcrip-tion factor
AtNAC1, promoting its degradation (Xie et al., 2000,2002). Although
tomato NAC1 is not orthologous to AtNAC1,which regulates
Arabidopsis root hair development through theauxin signaling (Xie
et al., 2000; Selth et al., 2005), it is possiblethat tomato NAC1
can be ubiquitinated via a similar RING-containing ubiquitin
ligase. Thus, we investigated whether aSINAT5-like ubiquitin ligase
in tomato can target NAC1 to pro-mote its degradation. We applied a
BLAST search for SINAT5homologs in the tomato genome database
(http://solge-nomics.net/) and found six tomato genes showing
significanthomology with SINAT5. The full-length cDNAs of these
sixSINAT5-like genes were cloned and named SINA1-6. Thededuced
SINA1-6 proteins share 78.6% identity at the aminoacid level,
containing a highly conserved RING domain, a typicalSINA-specific
Zn-finger domain and an SBD (Hu & Fearon,1999) (Fig. S1). To
test the possible direct interaction betweenSINA1-6 and NAC1, which
reflects an enzyme-substrate rela-tionship, we carried out a Y2H
assay in which SINA1-6 wereexpressed as prey and NAC11-260
(containing the N-terminal260 amino acids of NAC1; the full-length
NAC1 exhibitsautoactivation in yeast (Selth et al., 2005)) was
expressed as bait.Protein–protein interaction in Y2H was assessed
on X-Gal-containing medium and medium lacking leucine. As shown
inFig. 1(a), only yeast cells containing SINA3 and
NAC11-260exhibited blue coloration on the X-Gal plate, despite
normal pro-tein accumulation for all combinations of NAC11-260 and
SINAs.However, these yeast cells failed to grow on medium
lackingleucine (Fig. S2), suggesting there might be a weak
interactionbetween NAC1 and SINA3 when expressed in yeast.
New Phytologist (2016) 211: 138–148 � 2016 The AuthorsNew
Phytologist� 2016 New Phytologist Trustwww.newphytologist.com
Research
NewPhytologist140
http://solgenomics.net/http://solgenomics.net/
-
The apparent weak interaction between SINA3 and NAC1 inthe Y2H
assay led us to determine the possible in vivo interactionof NAC1
with SINA3 in plant cells using the coIP assay estab-lished in N.
benthamiana by our laboratory (Huang et al., 2013).To this end,
Agrobacteria containing the epitope-tagged full-length NAC1
(NAC1-FLAG) and SINA1-6-HA constructs, con-trolled by the
cauliflower mosaic virus (CaMV) 35S promoter,were infiltrated into
N. benthamiana leaves for transient expres-sion. To prevent NAC1
degradation, the proteasome inhibitorMG132 was added to the
Agrobacterium inoculum harboringappropriate constructs including a
vector control. As shown inFig. 1(b), NAC1 protein accumulated to a
considerable level inN. benthamiana leaves in the presence of
MG132. After proteinextraction and immunoprecipitation with the
a-HA antibodymatrix, the immunoprecipitated protein complex was
verified bywestern blotting using the a-FLAG antibody. The
NAC1-FLAG
protein was detected in the a-HA
antibody-immunoprecipitatedcomplex from the leaf tissue expressing
NAC1-FLAG andSINA3-HA (Fig. 1b), but not in the immunoprecipitated
com-plex from the leaf tissue expressing NAC1-FLAG and any
otherSINA-HAs or the vector control. This indicates that
NAC1specifically interacts with SINA3 in plant cells and suggests
thatSINA3 could be the ubiquitin E3 ligase responsible for
NAC1ubiquitination.
NAC1 and SINA3 interact in the nucleus
Next, we used confocal microscopy and BiFC (Walter et al.,2004;
Kanaoka et al., 2008) to determine where NAC1 andSINA3 are
localized within the plant cell and where NAC1-SINA3 complex
formation occurs. As shown in Fig. 2(a), underoverexpression
conditions in the presence of MG132 to preventNAC1 degradation,
NAC1-GFP was exclusively localized in thenucleus, whereas SINA3-GFP
was observed in both cytoplasmand nucleus. It is notable that,
unlike free GFP, the SINA3-GFPwas not evenly distributed in the
cytoplasm. To determine thelocalization of NAC1–SINA3 interaction,
NAC1, SINA3 andSINA31-181, the N-terminal 181-amino-acid region of
SINA3lacking the SBD, were each cloned into BiFC vectors (Walteret
al., 2004). The resulting constructs were expressed inN.
benthamiana leaves via Agrobacterium-mediated transientexpression
in the presence of MG132 and the BiFC analysis wasperformed 2 d
after agrobacterial infiltration. As shown inFig. 2(b), a strong
YFP signal was observed when NAC1-NYFPwas coexpressed with
SINA3-CYFP, but not with the controlSINA31-181-CYFP, which lacks
the SBD, and was limited in thenucleus of the N. benthamiana cells,
suggesting that NAC1 inter-acts with SINA3 in the nucleus.
SINA3 possesses ubiquitin ligase activity and ubiquitinatesNAC1
in vitro
The specific in planta interaction of NAC1 with SINA3
suggeststhat SINA3 may directly ubiquitinate the NAC1 substrate.
Toverify this notion, we first determined that the ubiquitin
ligaseactivity of SINA3 was capable of self-ubiquitination in the
pres-ence of ubiquitin E1 and E2 enzymes. We conducted the in
vitroubiquitination assay (Abramovitch et al., 2006) using
recombi-nant E1 (GST-AtUBA1) and E2 (GST-AtUBC8)
enzymes,FLAG-tagged ubiquitin (FLAG-Ub), and MBP-SINA3.
Self-ubiquitination of SINA3 was observed when FLAG-Ub, E1, andE2
were present (Fig. 3A, lane 1), but not in any control reactionin
which any one of the necessary components was missing(Fig. 3a upper
panel, lanes 2–5). Thus, SINA3 is a functionalubiquitin ligase.
To define NAC1 as a substrate of the SINA3 ubiquitin ligase,the
recombinant MBP-NAC1-HA protein was included in thein vitro
ubiquitination reaction mixture described earlier. Afterincubation,
the ubquitination reaction mixture was immunopre-cipitated with the
a-HA antibody matrix to purify the substrateMBP-NAC1-HA. The
purified MBP-NAC1-HA was subjectedto western blotting analysis
using a-FLAG or a-HA antibody to
Fig. 1 NAC1 interacts with a tomato SEVEN IN ABSENTIA (SINA)
ubiquitinligase. (a) NAC1 interacts with SINA3 in the LexA-based
yeast two- assay.The N-terminal 260 amino acids (NAC11-260), which
did not exhibit self-activation, were expressed as bait and SINA1-6
were expressed as prey.Blue yeast colonies grown on the
X-Gal-containing medium indicate theinteraction between NAC1 and
SINA3. The expression and accumulationof NAC11-260 and SINA
proteins in yeast were verified by western blottingusing a-LexA and
a-HA antibody, respectively (lower panel). (b) In vivointeraction
of NAC1 with SINA3 determined by
coimmunoprecipitation.Agrobacterium tumefaciens GV2260 strains
containing the cauliflowermosaic virus 35S promoter-driven
epitope-tagged SINA1-6 constructs(SINA1-6-HA), NAC1 (NAC1-FLAG)
construct or empty vector weresyringe-infiltrated into Nicotiana
benthamiana leaves at a concentration ofOD600 = 0.4. A. tumefaciens
containing the empty vector was used as anegative control. MG132
(100 lM) was coinjected with theAgrobacterium suspension to prevent
the degradation of NAC1-FLAGprotein. Two days after Agrobacterium
infiltration, proteins were extractedfor immunoprecipitation (IP)
with a-HA affinity matrix, followed bywestern blotting (WB) using
the a-FLAG antibody to determine theassociation of NAC1 with SINA3.
The presence of NAC1 protein (indicatedby an arrow in the bottom
right) in the immunoprecipitated complex ofNAC1 coexpressed with
SINA3 suggests specific interaction betweenthem. The asterisk
indicates a nonspecific cross-reaction band detected bythe
antibody.
� 2016 The AuthorsNew Phytologist� 2016 New Phytologist
Trust
New Phytologist (2016) 211: 138–148www.newphytologist.com
NewPhytologist Research 141
-
detect the presence of FLAG-Ub, MBP-NAC1-HA
andFLAG-Ub-MBP-NAC1-HA protein conjugates. As expected,
aubiquitin-associated ladder-like smear, indicating the presence
ofpolyubiquitination of both MBP-SINA3 (self-ubiquitination)and
MBP-NAC1-HA, was detected by the a-FLAG antibody(Fig. 3b upper
panel, lane 1). The identity of polyubiquitinatedMBP-NAC1-HA was
further verified by the a-HA antibody(Fig. 3b lower panel, lane 1),
suggesting that SINA3 can ubiquiti-nate NAC1 in vitro. Thus, we
conclude SINA3 is a ubiquitinligase capable of ubiquitinating
NAC1.
SINA3 promotes NAC1 degradation in planta
viapolyubiquitination
Next we sought to determine whether SINA3 can promoteNAC1
degradation in vivo through ubiquitination. As NAC1 israpidly
degraded in tomato and cannot be detected by westernblotting
without the presence of MG132 (Huang et al., 2013),we
heterogeneously expressed NAC1 in N. benthamiana, inwhich the
Agrobacterium-mediated transiently expressed NAC1accumulated to
certain level that can be detected by western
(a) (b)
GFP
Bar = 20 μm
NAC1-NYFP/
SINA3-CYFP
NAC1-NYFP/
SINA1-181-CYFP3
NYFP/SINA3-
CYFP
YFPDAPI DAPIGFP
SINA3-
GFP
NAC1-
GFP
DIC
DIC
DIC DIC
DIC
DIC
MergeMerge
Fig. 2 NAC1 and SEVEN IN ABSENTIA3 (SINA3) interact in the
nucleus. (a) Subcellular localization of NAC1 and SINA3.
Agrobacterium tumefaciensGV2260 strains harboring the cauliflower
mosaic virus (CaMV) 35S promoter-driven NAC1-GFP, SINA3-GFP or free
green fluorescent protein (GFP) wereinfiltrated into Nicotiana
benthamiana leaves at inoculums of OD600 = 0.4. Two days after
agroinfiltration, the infiltrated leaf tissue was subjected
toconfocal microscopy, which indicates that the NAC1-GFP is
exclusively localized in the nucleus, whereas the SINA3-GFP is
localized in both cytoplasm andnucleus. Preceding
40,6-diamidino-2-phenylindole (DAPI) staining of the leaf tissue
verifies the nucleus. Differential interference contrast (DIC)
images ofthe same view are aligned underneath the GFP signal
images. (b) NAC1 interacts with SINA3 in the nucleus. The CaMV 35S
promoter-driven NAC1-NYFPand SINA3-CYFP constructs were coexpressed
in N. benthamiana leaves as in (a). After DAPI staining of leaf
tissue to locate the nucleus, epidermal celllayers were examined
using a confocal microscope to capture the yellow fluorescent
protein (YFP) signal resulting from interaction between NAC1
andSINA3. Coexpression of NAC1-NYFP with SINA31-181-CYFP or NYFP
with SINA3-CYFP did not result in production of YFP signal and
served as negativecontrols, in which no restored YFP signal was
observed. DIC images of the same view are aligned underneath the
YFP signal images.
New Phytologist (2016) 211: 138–148 � 2016 The AuthorsNew
Phytologist� 2016 New Phytologist Trustwww.newphytologist.com
Research
NewPhytologist142
-
blotting (Fig. 4a). We found that the NAC1 level was
dramati-cally attenuated when coexpressed with SINA3, while its
accu-mulation was not significantly affected when coexpressed
withthe vector control or the nonfunctional mutant SINA3C72S
(Fig. 4a), in which the conserved Cys was substituted with a
Serin the RING domain (Den Herder et al., 2012). This
resultindicates that SINA3 promotes NAC1 degradation in vivo.
Ingeneral, the 26S proteasome-mediated degradation is dependenton
polyubiquitination of the target protein. Thus, we nextsought to
examine the potential ubiquitination of NAC1 bySINA3 in vivo.
Again, NAC1-FLAG and HA-tagged ubiquitinwere coexpressed in N.
benthamiana leaves with or without thepresence of SINA3-HA, and the
possible ubiquitination ofNAC1-HA triggered by SINA3 was
investigated. In order toobserve the ubiquitinated NAC1 protein,
the proteasome-specific inhibitor MG-132 was included in the
agrobacterialinoculum to prevent NAC1 degradation. Total protein
wasextracted at 36 h post agroinfiltration, followed by western
blot-ting using a-FLAG antibody to detect NAC1 protein. Asshown in
Fig. 4(b), the polyubiquitinated NAC1-HA moieties,indicated as the
smear banding pattern above the NAC1-HA,were detected in the
presence of SINA3, suggesting that SINA3may ubiquitinate NAC1 in
vivo. It is notable that in theabsence of SINA3, a slow-migrated
NAC1 form was observed(indicated by the asterisk in Fig. 4b). The
identity of thisNAC1 band was not determined. Significantly, when
SINA3was coexpressed, this slow-migrated form was abolished and
theNAC1 was heavily polyubiquitinated instead. Taken together,these
results suggest that SINA3 is a ubiquitin E3 ligase
promoting the ubiquitination-mediated degradation of NAC1in
plant cells.
In contrast to up-regulation of the NAC1 gene, theexpression of
the SINA3 gene is down-regulated during thedefense response to
Pseudomonas infection
Given the fact that NAC1 plays an important role in the
defenseresponse and is subjected to UPS-mediated degradation,
up-regulation of NAC1 gene transcription during the defense
responsecould be a strategy used by plants to produce more NAC1
proteinto compensate for its degradation by ubiquitin ligases
(Huang et al.,2013). Alternatively, plants might adopt other
mechanisms to inter-fere with the ubiquitination of NAC1 protein,
such as repressingtranscription of the cognate ubiquitin ligase
genes. To test the latterhypothesis, we determined SINA3 mRNA
abundance during theresponse to Pseudomonas syringae pv. tomato
strain DC3000(PstDC3000) infection. Following the experimental
regime used inour previous studies on NAC1(Huang et al., 2013), we
examinedSINA3 mRNA abundance in three different interactions
betweentomato and Pst: the R-mediated immune interaction in which
resis-tant RG-PtoR plants (expressing the resistance gene Prf) were
inocu-lated with PstDC3000; the disease interaction in which
susceptibleRG-prf3 plants (containing a 1 kb deletion in the Prf
gene;Salmeron et al., 1996) were inoculated with PstDC3000; and
thepathogen-associated molecular pattern-triggered immune
interac-tion in which susceptible RG-prf3 plants were inoculated
with thenonpathogenic PstDC3000 hrcC mutant strain (Deng et al.,
1998).RG-prf3 plants infiltrated with 10 mM MgCl2 served as a
mock
Fig. 3 SEVEN IN ABSENTIA3 (SINA3)ubiquitinates NAC1 in vitro.
(a) The ubiquitinE3 activity of SINA3. Polyubiquitination ofSINA3
(E3 ligase) was observed in thereaction in the presence of
recombinant E1,E2 and FLAG-Ub (lane 1), but not in anycontrol
reaction in which any of thenecessary components was missing (lanes
2–5). Polyubiquitinated forms of SINA3, causedby
self-ubiquitination of SINA3, verify theubiquitin ligase activity
of SINA3. Coomassiestaining of the western blotting (WB)indicates
an equal amount of SINA3 presentin the reactions (lower panel).
(b)Polyubiquitination of NAC1 by SINA3 in thepresence of
recombinant E1, E2, maltosebinding protein (MBP)-SINA3,
MBP-NAC1-hemagglutinin (HA) and FLAG-Ub (lane 1).The in vitro
ubiquitination reaction mixturewas immunoprecipitated with a-HA
antibodymatrix to purify the MBP-NAC1-HAsubstrate, followed by WB
using a-FLAGantibody (top panel) or a-HA antibody(lower panel) to
determine allpolyubiquitination forms (including
self-ubiquitination of SINA3, which representsthe majority of the
polyubiquitinated protein)and the presence of polyubiquitinated
MBP-NAC1-HA protein, respectively.
� 2016 The AuthorsNew Phytologist� 2016 New Phytologist
Trust
New Phytologist (2016) 211: 138–148www.newphytologist.com
NewPhytologist Research 143
-
control. By real-time reverse transcription polymerase chain
reac-tion analysis, we monitored the transcript abundance of the
SINA3and NAC1 genes at different time points after bacterial
infiltrationto determine the correlation between the expression
patterns ofthese two genes in response to pathogen infection. As
shown inFig. 5, NAC1 mRNA abundance was increased in all three
interac-tions, which was consistent with our previous publication
(Huanget al., 2013), whereas the expression of the SINA3 mRNA
wasreciprocally repressed. Significantly, the down-regulation
pattern ofthe SINA3 gene was inversely correlated with the
up-regulation pat-tern of the NAC1 gene in the three different
tomato–Pst interac-tions. Taken together, our results suggest that
tomato plants haveevolved a complex mechanism to regulate the
defense-relatedNAC1 transcription factor in response to Pseudomonas
infection:up-regulation of the NAC1 gene to produce more NAC1
proteinand down-regulation of the SINA3 gene encoding the
ubiquitinligase to compensate for NAC1 degradation.
Overexpression of SINA3 represses the R protein-mediatedHR cell
death in N. benthamiana
We next sought to determine the biological significance of
theSINA3 ubiquitin E3 ligase in plant defense signaling. We
firstexamined the effect of SINA3 on HR cell death signaling
medi-ated by the tomato resistance protein Prf, which confers
resis-tance to Pst (Salmeron et al., 1996). The PrfD1416V mutant is
anautoactive form of Prf and can trigger the HR cell death
whentransiently expressed in N. benthamiana leaves (Du et al.,
2012).We coexpressed SINA3 and PrfD1416V in N. benthamiana
leaves via Agrobacterium-mediated transient expression at a 4
:1(SINA3:PrfD1416V) inoculum ratio. We found that cell
deathtriggered by PrfD1416V was abolished in the presence of
SINA3(Fig. 6a), suggesting that SINA3 negatively regulates the HR
celldeath signaling. As SINA3 is a ubiquitin ligase, one possible
rea-son for this suppression of cell death is that SINA3
ubiquitinatesPrfD1416V, resulting in its degradation. To test this
possibility, weexamined PrfD1416V accumulation in planta with or
without thepresence of SINA3. PrfD1416V causes a strong cell death
reactionthat might trigger nonspecific protein degradation, so we
coex-pressed PrfD1416V with SINA3 at a slightly higher
concentrationof inoculum, OD600 = 0.3. Western blotting analysis
indicatedthat coexpression of SINA3 with PrfD1416V did not
affectPrfD1416V accumulation (Fig. 6a), suggesting that SINA3
doesnot lead to PrfD1416V protein degradation.
We next asked whether the cell death suppression activity
ofSINA3 is specific to Prf-mediated HR cell death signaling,
orwhether SINA3 acts as a general negative regulator for HR
celldeath signaling. We assessed the ability of SINA3 to
interferewith HR cell death triggered by two other autoactive R
proteins,Rpi-blb1D475V and RxD460V, both of which cause HR cell
deathwhen overexpressed in N. benthamiana leaves (Bendahmaneet al.,
2002; van Ooijen et al., 2008). SINA3 was coexpressedwith
Rpi-blb1D475V or RxD460V in N. benthamiana leaves at thesame 4 : 1
Agrobacterium inoculum ratio as used for PrfD1416V. Inaround 50% of
experimental repetitions, we found that Rpi-blb1D475V- or
RxD460V-triggered cell death was suppressed byoverexpression of
SINA3, and SINA3 indeed does not triggerRpi-blb1D475V or RxD460V
degradation (Fig. 6b,c). Representa-tive leaf tissue showing
SINA3-mediated cell death suppression isshown in Fig. 6(b). We note
that the cell death suppression activ-ity of SINA3 for
Rpi-blb1D475V- or RxD460V-induced HR wasless effective than that
observed for the suppression of PrfD1416V-induced HR, which is
probably a result of extremely strong celldeath elicited by
overexpression of Rpi-blb1D475V or RxD460V
(van Ooijen et al., 2008). The RxD460V protein was not
detectedwhen expressed with the empty vector control (Fig. 6c),
presum-ably as a result of the nonspecific protein degradation
caused bysuch extremely strong cell death.
Discussion
As NAC transcription factors are one of the largest families
ofplant-specific transcription factors and they have significant
rolesin diverse physiological processes, it is not surprising to us
thatthe regulation of NAC transcription factors is complex
andoccurs at multiple levels. First, NAC transcription factors can
beregulated at the transcriptional level: up- or down-regulated
inresponse to internal or external stimuli, which is the case
fornumerous NAC transcription factors (Olsen et al., 2005;
Puraniket al., 2012). Second, a few Arabidopsis NAC transcription
fac-tors, including CUC1/2 and AtNAC1, are regulated at the
post-transcriptional level via miRNA-mediated cleavage (Olsen et
al.,2005; Puranik et al., 2012). Third, several NAC
transcriptionfactors are subjected to post-translational
modifications, such asphosphorylation of Arabidopsis NTL6 and rice
OsNAC2 by the
Fig. 4 SEVEN IN ABSENTIA3 (SINA3) promotes NAC1 degradation
inplanta via polyubiquitination. (a) Agrobacterium tumefaciens
GV2260strains harboring the cauliflower mosaic virus (CaMV) 35S
promoter-driven FLAG-tagged NAC1 (NAC1-FLAG), in combination with
the HA-tagged SINA3 (SINA3-HA), SINA3C72S mutant (SINA3C72S-HA), or
emptyvector, were infiltrated into Nicotiana benthamiana leaves at
aconcentration of OD600 = 0.3. Leaf tissue was harvested at 32 h
afterinfiltration for western blotting (WB) using the a-FLAG or
a-HA antibody.(b) Agrobacterium tumefaciens GV2260 strains
harboring the CaMV 35Spromoter-driven constructs of epitope-tagged
ubiquitin (HA-Ub) or NAC1(NAC1-FLAG) were infiltrated into N.
benthamiana leaves at aconcentration of OD600 = 0.4. A. tumefaciens
containing the empty vector(Vector) was used as a control. MG132
(100 lM) was coinjected with theAgrobacterium suspension to prevent
the degradation of NAC1 protein.Thirty-six hours after
Agrobacterium infiltration, proteins were extractedfor WB assay
using a-FLAG antibody to determine NAC1-associatedpolyubiquitin
chain, which appears as a smear. The asterisk indicates
anunidentified modification form of NAC1.
New Phytologist (2016) 211: 138–148 � 2016 The AuthorsNew
Phytologist� 2016 New Phytologist Trustwww.newphytologist.com
Research
NewPhytologist144
-
SnRK2.8 kinase and an as-yet-unidentified kinase (Kaneda et
al.,2009; Kim et al., 2012), respectively, and ubiquitination
ofArabidopsis AtNAC1 by the SINAT5 ubiquitin ligase (Xie et
al.,2002). We previously found that the tomato NAC1
transcriptionfactor is regulated at both the transcriptional and
post-translational levels, as manifested by increased NAC1
mRNAabundance in response to Pseudomonas infection and the
ubiqui-tination of NAC1 protein (Huang et al., 2013). In this work,
wesought to identify a ubiquitin ligase that regulates NAC1
proteinaccumulation. We examined six tomato SINA ubiquitin
ligases(SINA1-6) and determined that SINA3 specifically interacts
withNAC1 in plant cells. We also found that SINA3 ubiquitinatesNAC1
in vitro and promotes NAC1 degradation via polyubiqui-tintion in
vivo. In addition, we demonstrated that SINA3 inter-feres with HR
cell death signaling mediated by multiple Rproteins. Our results
support the hypothesis that SINA3 ubiqui-tin ligase negatively
regulates cell death signaling by promoting
the ubiquitination-mediated degradation of
defense-relatedproteins.
Among the six highly homologous tomato SINAs
(SINA1-6)identified, SINA3 interacts with and ubiquitinates the
defense-related NAC1 transcription factor in Y2H assay. Although
theSINA3–NAC1 interaction was found to be weak in yeast (wecould
only detect the interaction on X-Gal-dependent, but
notleucine-dependent, assay, which in fact suggests the
C-terminal261–301 amino acid region of NAC1 is required for the
interac-tion), it was also further verified by coIP assay in
planta. Theubiquitination of NAC1 by SINA3 was determined by
polyubiq-uitination of NAC1 when ubiquitin E1, E2, SINA3 and
NAC1were incubated with the ubiquitin molecule. It is
generallythought that polyubiquitination of a substrate protein
results inproteasomal proteolysis, and thus coexpression of the
ubiquitinligase with the substrate may promote the degradation of
the sub-strate. This notion was substantiated by the degradation of
the
SINA3
Rel
ativ
eex
pres
sion
ratio
prf3/ mock
prf3/ hrcC
prf3/ DC3000
PtoR/ DC3000
*
*
NAC1
evitaleR
noisserpxeoitar
prf3/ mock
prf3/ hrcC
prf3/ DC3000
PtoR/ DC3000
*
** *
*
*
*
*
** ******
****
****
(a) (b)
Fig. 5 Down-regulation of the SEVEN IN ABSENTIA3 (SINA3) gene by
Pseudomonas infection. (a, b) Up-regulation of the NAC1 gene (a)
but down-regulation of the SINA3 gene (b) during different
plant–pathogen interactions. Resistant RG-PtoR or susceptible
RG-prf3 tomato plants were inoculatedwith appropriate Pseudomonas
syringae pv. tomato (Pst) DC3000 strains at an inoculum of 29 107
colony-forming units ml�1 or mock solution (10mMMgCl2). In all
cases, total RNA was isolated at the indicated time points after
Pst infiltration. The relative expression level of NAC1 or SINA3
genes wereanalyzed by real-time reverse transcription polymerase
chain reaction using the gene-specific primers. The expression of
the tomato EF1-a gene served asan internal control for
normalization. Values are means� SE of three replicates. Asterisks
represent significant differences (**, P < 0.001; *, P <
0.05, bytwo-tailed Student’s t-test) in NAC1 or SINA3 gene
expression between 0 h and other time points after Pst or mock
treatment. The experiment wasrepeated three times with similar
results.
(a)
VectorSINA3-HA
Rpi-blb1D475V-HA RxD460V-HA
VectorSINA3-HAVectorSINA3-HA
PrfD1416V-FLAG
SINA3-HA
Rpi-blb1D475V-HA
kD
-100
-55
-35
-130
SINA3-HA
Prf D1416V-FLAG
kD
-250
-55
-35
SINA3-HA
RxD461V-HA
kD
-100
-55
-35
-130
(b) (c)
Fig. 6 SEVEN IN ABSENTIA3 (SINA3) represses hypersensitive
response (HR) cell death mediated by multiple resistance proteins
in Nicotiana benthamiana.Overexpression of SINA3 interferes with HR
cell death mediated by multiple R proteins. (a–c) Agrobacterium
tumefaciens GV2260 strains carrying theindicated constructs were
syringe-infiltrated into N. benthamiana leaves at OD600 = 0.2 for
Prf
D1416V-FLAG (a), Rpi-blb1D475V (b) or Rx D460V (c) andOD600 =
0.8 for SINA3-HA or vector control. All genes were expressed from
the 35S cauliflower mosaic virus (CaMV) promoter. Photographs were
taken 3 dafter Agrobacterium infiltration. The assay results
provided below indicate that SINA3 does not trigger degradation of
PrfD1416V, Rpi-blb1D475V or RxD460V.
� 2016 The AuthorsNew Phytologist� 2016 New Phytologist
Trust
New Phytologist (2016) 211: 138–148www.newphytologist.com
NewPhytologist Research 145
-
exogenously expressed NAC1 in the presence of coexpression
ofSINA3 (Fig. 4a), which is consistent with the in vivo
polyubiqui-tination of NAC1 triggered by SINA3 in N.
benthamiana(Fig. 4b). Taken together, our data suggest that SINA3
ubiquiti-nates NAC1 transcription factor to promote its
degradation. It isinteresting to note that, without coexpression of
SINA3, NAC1protein also accumulated as a slow-migrated form (Fig.
4b), rep-resenting an unidentified modification of NAC1. We
speculatethat this could be a monoubiquitinated and/or
phosphorylatedform of NAC1, a topic to be explored in future
experiments. Sig-nificantly, such modification was abolished when
SINA3 wasapplied and the main modification of NAC1 changed to
polyu-biquitination.
Our previous publication demonstrated that the defense-related
NAC1 transcription factor is fine-tuned at both transcrip-tional
and post-translational levels and is important for plantdisease
resistance (Huang et al., 2013). It is reasonable to specu-late
that under normal conditions without pathogen infection,plants need
to tightly control transcription factors like NAC1 toprevent
autoactivation of defense signaling. At the post-translational
level, a rapid turnover of NAC1 protein may help tomaintain the
signaling balance, while at the transcriptional level,the NAC1 gene
is expressed at a basal level to produce a limitedamount of NAC1
protein. Upon pathogen infection, plantscould employ mechanisms to
counteract the degradation ofNAC1 protein. One is rapid expression
of the NAC1 gene inorder to produce more protein, ideally
overcoming the degrada-tion of NAC1, while the other would be
interference with ubiq-uitination of NAC1 to prevent its
degradation, which can beachieved by transcriptional
down-regulation of the SINA3 ubiq-uitin ligase gene. Reduced mRNA
abundance of the SINA3 genein response to PstDC3000 infection
supports this hypothesis.Unfortunately, because of a lack of a
NAC1-specific antibody, wewere unable to determine the dynamic
change of ubiquitinationand degradation of native tomato NAC1
protein during thedefense response to Pseudomonas infection.
Generation of trans-genic tomato plants expressing epitope-tagged
NAC1-HA underthe control of a constitutive promoter (such as the
CaMV 35Spromoter instead of the stress-inducible native promoter)
willavoid protein accumulation as a result of transcriptional
induc-tion of the NAC1 gene and should enable us to address
dynamicregulation of NAC1 protein levels in future work.
Given the fact that NAC1 plays a positive role in the
plantdefense response (Huang et al., 2013) and SINA3
ubiquitinatesNAC1 for promoting its degradation (Figs 3, 4), it is
logical tohypothesize that SINA3 functions as a negative regulator
in plantdefense signaling. This was substantiated by the
down-regulationof SINA3 gene expression in response to Pseudomonas
infection(Fig. 5) and interference of overexpressed SINA3 with HR
celldeath triggered by multiple R proteins (Fig. 6). However, it
ispossible that, besides targeting the NAC1 transcription
factor,SINA3 also ubiquitinates other positive defense regulator(s)
topromote its degradation, thereby repressing HR cell death
signal-ing during the defense response. In fact, the
SINA3–NAC1interaction is exclusively restricted in the nucleus
(Fig. 2b), sug-gesting that SINA3 ubiquitinates NAC1 in the nucleus
for
proteasome-mediated degradation. Additionally, NAC1 is
exclu-sively localized in the nucleus, whereas SINA3 is localized
inboth the nucleus and cytoplasm. Therefore, it is possible that
thecytoplasm-localized SINA3 also targets other unknown
factors,thereby promoting their degradation in the cytoplasm.
Thus,identification of additional SINA3 ubiquitin ligase substrates
willbe helpful to elucidate the mechanistic basis of
SINA3-mediatedregulation in defense response.
It is interesting to note that all plant SINA ubiquitin
ligasesidentified to date from four plant species (SINAT5
fromArabidopsis, SINA4 from Lotus japonicus, OsDIS1 from rice
andSINA3 from tomato) function as negative regulators of
certainphysiological processes: the Arabidopsis SINAT5 negatively
con-trols lateral root growth by targeting the transcription
factorAtNAC1 essential for the auxin-mediated lateral root
develop-ment (Xie et al., 2002); the L. japonicus SINA4 negatively
regu-lates Sinorhizobium infection (Den Herder et al., 2012) and
isspeculated to exert a negative effect on nodulation as it
apparentlytargets the symbiosis receptor-like kinase (SYMRK), a
positiveregulator in symbiotic signal transduction, for promoting
itsdegradation (Den Herder et al., 2012); the rice OsDIS1 plays
anegative role in drought stress tolerance, presumably by
targetingthe tubulin complex-related kinase OsNek6 despite lack of
evi-dence for the ubiquitination of OsNsk6 by OsDIS1(Ning et
al.,2011a); and in this study we demonstrate that SINA3, and
possi-bly other SINA isoforms, acts as a negative regulator for
celldeath signaling. Therefore, it seems that plant SINA
ubiquitinligases generally act as negative regulators by targeting
impor-tant positive components in certain signaling pathways. In
par-ticular, we found in this study that SINA3 targets a
defensepositive regulator SlNAC1. As SINA3 interferes with HR
celldeath triggered by multiple R proteins without affecting
theiraccumulation level, it is likely that SINA3 also targets a
con-served cell death positive regulator(s), functioning
downstreamof the R protein at a convergence point of HR cell death
sig-naling, for ubiquitination and degradation. It will be
interest-ing to test whether SINA3 can ubiquitinate the known
celldeath regulators (e.g. MAPKKKa; del Pozo et al., 2004) and/or
explore other putative ubiquitination targets of SINA3 usinga
biochemical approach such as Y2H screening, which will bea subject
of our future research.
Acknowledgements
We thank Hailey Youngling, Anthony Trakas and MarcelinaStarok
for technical assistance and Dr Allan Caplan for criticalreading of
the manuscript. This work was supported in part bygrants from the
Agriculture and Food Research Initiative Competi-tive Grants
Program from the USDA (no. 2010-6511-42056), theIdaho State
Department of Agriculture and the Idaho Potato Com-mission to F.X.,
the Idaho State Department of Agriculture andthe Idaho Potato
Commission to J.C.K., the Advanced Program ofDoctoral Fund of
Ministry of Education of China(20110181130009), the National Basic
Research Program ofChina (973 Program) (no. 2011CB100401), and the
NationalNatural Science Foundation of China (no. 31461143008) to
Y.L.
New Phytologist (2016) 211: 138–148 � 2016 The AuthorsNew
Phytologist� 2016 New Phytologist Trustwww.newphytologist.com
Research
NewPhytologist146
-
Author contributions
M.M., X.N., J.K. and X.D. performed the research; Y.L. andF.X.
designed the research; J.A. and T.P.D. contributed newreagents;
Y.L., J.C.K. and F.X. analyzed the data; and Y.L. andF.X. wrote the
paper.
References
Abramovitch RB, Janjusevic R, Stebbins CE, Martin GB. 2006. Type
III
effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity
to suppress plant
cell death and immunity. Proceedings of the National Academy of
Sciences, USA103: 2851–2856.
Bendahmane A, Farnham G, Moffett P, Baulcombe DC. 2002.
Constitutive
gain-of-function mutants in a nucleotide binding site-leucine
rich repeat
protein encoded at the Rx locus of potato. Plant Journal 32:
195–204.Carthew RW, Rubin GM. 1990. seven in absentia, a gene
required for
specification of R7 cell fate in the Drosophila eye. Cell 63:
561–577.Delessert C, Kazan K, Wilson IW, Van Der Straeten D,
Manners J, Dennis ES,
Dolferus R. 2005. The transcription factor ATAF2 represses the
expression of
pathogenesis-related genes in Arabidopsis. Plant Journal 43:
745–757.Den Herder G, De Keyser A, De Rycke R, Rombauts S, Van de
Velde W,
Clemente MR, Verplancke C, Mergaert P, Kondorosi E, Holsters M
et al.2008. Seven in absentia proteins affect plant growth and
nodulation in
Medicago truncatula. Plant Physiology 148: 369–382.Den Herder G,
Yoshida S, Antolin-Llovera M, Ried MK, Parniske M. 2012.
Lotus japonicus E3 ligase SEVEN IN ABSENTIA4 destabilizes the
symbiosisreceptor-like kinase SYMRK and negatively regulates
rhizobial infection. PlantCell 24: 1691–1707.
Deng WL, Preston G, Collmer A, Chang CJ, Huang HC. 1998.
Characterization of the hrpC and hrpRS operons of Pseudomonas
syringaepathovars syringae, tomato, and glycinea and analysis of
the ability of hrpF,hrpG, hrcC, hrpT, and hrpV mutants to elicit
the hypersensitive response and
disease in plants. Journal of Bacteriology 180: 4523–4531.Du X,
Miao M, Ma X, Liu Y, Kuhl JC, Martin GB, Xiao F. 2012. Plant
programmed cell death caused by an autoactive form of Prf is
suppressed by co-
expression of the Prf LRR domain.Molecular Plant 5:
1058–1067.Golemis EA, Serebriiskii I, Finley RL Jr, Kolonin MG,
Gyuris J, Brent R. 2001.
Interaction trap/two-hybrid system to identify interacting
proteins. CurrentProtocols in Protein Science 14:
19.2:19.2.1–19.2.40.
Harper JW, Schulman BA. 2006. Structural complexity in ubiquitin
recognition.
Cell 124: 1133–1136.House CM, Frew IJ, Huang HL, Wiche G,
Traficante N, Nice E, Catimel B,
Bowtell DD. 2003. A binding motif for Siah ubiquitin ligase.
Proceedings of theNational Academy of Sciences, USA 100:
3101–3106.
Hu G, Fearon ER. 1999. Siah-1 N-terminal RING domain is
required
for proteolysis function, and C-terminal sequences regulate
oligomerization
and binding to target proteins.Molecular and Cellular Biology
19:724–732.
Huang W, Miao M, Kud J, Niu X, Ouyang B, Zhang J, Ye Z, Kuhl JC,
Liu Y,
Xiao F. 2013. SlNAC1, a stress-related transcription factor, is
fine-tuned on
both the transcriptional and the post-translational level. New
Phytologist 197:1214–1224.
Kanaoka MM, Pillitteri LJ, Fujii H, Yoshida Y, Bogenschutz NL,
Takabayashi
J, Zhu JK, Torii KU. 2008. SCREAM/ICE1 and SCREAM2 specify three
cell-
state transitional steps leading to Arabidopsis stomatal
differentiation. PlantCell 20: 1775–1785.
Kaneda T, Taga Y, Takai R, Iwano M, Matsui H, Takayama S, Isogai
A, Che
FS. 2009. The transcription factor OsNAC4 is a key positive
regulator of plant
hypersensitive cell death. EMBO Journal 28: 926–936.Kim MJ, Park
MJ, Seo PJ, Song JS, Kim HJ, Park CM. 2012. Controlled
nuclear import of the transcription factor NTL6 reveals a
cytoplasmic role
of SnRK2.8 in the drought-stress response. Biochemical Journal
448: 353–363.
Ning Y, Jantasuriyarat C, Zhao Q, Zhang H, Chen S, Liu J, Liu L,
Tang S, Park
CH, Wang X et al. 2011a. The SINA E3 ligase OsDIS1 negatively
regulatesdrought response in rice. Plant Physiology 157:
242–255.
Ning Y, Xie Q, Wang GL. 2011b.OsDIS1-mediated stress response
pathway in
rice. Plant Signaling & Behavior 6: 1684–1686.Olsen AN,
Ernst HA, Leggio LL, Skriver K. 2005. NAC transcription
factors:
structurally distinct, functionally diverse. Trends in Plant
Science 10: 79–87.van Ooijen G, Mayr G, Kasiem MM, Albrecht M,
Cornelissen BJ, Takken FL.
2008. Structure-function analysis of the NB-ARC domain of plant
disease
resistance proteins. Journal of Experimental Botany 59:
1383–1397.Park BS, Eo HJ, Jang IC, Kang HG, Song JT, Seo HS. 2010.
Ubiquitination of
LHY by SINAT5 regulates flowering time and is inhibited by
DET1.
Biochemical and Biophysical Research Communications 398:
242–246.del Pozo O, Pedley KF, Martin GB. 2004.MAPKKKa is a
positive regulator of
cell death associated with both plant immunity and disease. EMBO
Journal 23:3072–3082.
Puranik S, Sahu PP, Srivastava PS, Prasad M. 2012. NAC proteins:
regulation
and role in stress tolerance. Trends in Plant Science 17:
369–381.Ravid T, Hochstrasser M. 2008.Diversity of degradation
signals in the ubiquitin–proteasome system. Nature Reviews
Molecular Cell Biology 9: 679–690.
Sadanandom A, Bailey M, Ewan R, Lee J, Nelis S. 2012. The
ubiquitin–proteasome system: central modifier of plant signalling.
New Phytologist 196:13–28.
Salmeron JM, Oldroyd GED, Rommens CMT, Scofield SR, Kim H-S,
Lavelle
DT, Dahlbeck D, Staskawicz BJ. 1996. Tomato Prf is a member of
theleucine-rich repeat class of plant disease resistance genes and
lies embedded
within the Pto kinase gene cluster. Cell 86: 123–133.Selth LA,
Dogra SC, Rasheed MS, Healy H, Randles JW, Rezaian MA. 2005. A
NAC domain protein interacts with tomato leaf curl virus
replication accessory
protein and enhances viral replication. Plant Cell 17:
311–325.Walter M, Chaban C, Schutze K, Batistic O, Weckermann K,
Nake C, Blazevic
D, Grefen C, Schumacher K, Oecking C et al. 2004. Visualization
of proteininteractions in living plant cells using bimolecular
fluorescence
complementation. Plant Journal 40: 428–438.Wang M, Jin Y, Fu J,
Zhu Y, Zheng J, Hu J, Wang G. 2008. Genome-wide
analysis of SINA family in plants and their phylogenetic
relationships. DNASequence 19: 206–216.
Wang X, Basnayake BM, Zhang H, Li G, Li W, Virk N, Mengiste T,
Song F.
2009. The Arabidopsis ATAF1, a NAC transcription factor, is a
negative
regulator of defense responses against necrotrophic fungal and
bacterial
pathogens.Molecular Plant–Microbe Interactions 22:
1227–1238.Welsch R, Maass D, Voegel T, Dellapenna D, Beyer P. 2007.
Transcription
factor RAP2.2 and its interacting partner SINAT2: stable
elements in the
carotenogenesis of Arabidopsis leaves. Plant Physiology 145:
1073–1085.Wu Y, Deng Z, Lai J, Zhang Y, Yang C, Yin B, Zhao Q,
Zhang L, Li Y, Xie Q.
2009. Dual function of Arabidopsis ATAF1 in abiotic and biotic
stress
responses. Cell Research 19: 1279–1290.Xiao F, He P, Abramovitch
RB, Dawson JE, Nicholson LK, Sheen J, Martin
GB. 2007. The N-terminal region of Pseudomonas type III effector
AvrPtoBelicits Pto-dependent immunity and has two distinct
virulence determinants.
Plant Journal 52: 595–614.Xie Q, Frugis G, Colgan D, Chua NH.
2000. Arabidopsis NAC1 transduces
auxin signal downstream of TIR1 to promote lateral root
development. Genes& Development 14: 3024–3036.
Xie Q, Guo HS, Dallman G, Fang S, Weissman AM, Chua NH. 2002.
SINAT5
promotes ubiquitin-related degradation of NAC1 to attenuate
auxin signals.
Nature 419: 167–170.
Supporting Information
Additional supporting information may be found in the
onlineversion of this article.
Fig. S1 Alignment of six tomato SINA ubiquitin ligases.
� 2016 The AuthorsNew Phytologist� 2016 New Phytologist
Trust
New Phytologist (2016) 211: 138–148www.newphytologist.com
NewPhytologist Research 147
-
Fig. S2 A weak interaction between NAC1 and SINA3 identifiedby
the LexA-based Y2H assay.
Table S1 List of primers used in this study
Please note: Wiley Blackwell are not responsible for the
contentor functionality of any supporting information supplied by
theauthors. Any queries (other than missing material) should
bedirected to the New Phytologist Central Office.
New Phytologist is an electronic (online-only) journal owned by
the New Phytologist Trust, a not-for-profit organization
dedicatedto the promotion of plant science, facilitating projects
from symposia to free access for our Tansley reviews.
Regular papers, Letters, Research reviews, Rapid reports and
both Modelling/Theory and Methods papers are encouraged. We are
committed to rapid processing, from online submission through to
publication ‘as ready’ via Early View – our average timeto decision
is